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Conditional Gene Targeting Reveals Cell Type-Specific Roles of the Lysosomal Protease Cathepsin L in Mammary Tumor Progression

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Conditional Gene Targeting Reveals Cell Type-Specific Roles of the Lysosomal Protease Cathepsin L in Mammary Tumor Progression cancers Article Conditional Gene Targeting Reveals Cell Type-Specific Roles of the Lysosomal Protease Cathepsin L in Mammary Tumor Progression María Alejandra Parigiani 1,2, Anett Ketscher 1, Sylvia Timme 3,4,5, Peter Bronsert 3,4,5 , Manuel Schlimpert 2,6, Bernd Kammerer 6,7, Arnaud Jacquel 8,9,10, Paul Chaintreuil 8,9,10 and Thomas Reinheckel 1,7,11,* 1 Institute of Molecular Medicine and Cell Research, Faculty of Medicine, Albert-Ludwigs-University of Freiburg, Stefan Meier Str. 17, 79104 Freiburg, Germany; [email protected] (M.A.P.); [email protected] (A.K.) 2 Faculty of Biology, Albert-Ludwigs-University of Freiburg, Schaenzle Str. 1, 79104 Freiburg, Germany; [email protected] 3 Institute for Surgical Pathology, Medical Center-University of Freiburg, Breisacher Str. 115A, 79106 Freiburg, Germany; [email protected] (S.T.); [email protected] (P.B.) 4 Tumorbank Comprehensive Cancer Center Freiburg, Medical Center–University of Freiburg, 79106 Freiburg, Germany 5 Faculty of Medicine, Albert-Ludwigs-University of Freiburg, Breisacher Str. 153, 79110 Freiburg, Germany 6 Center for Biological Systems Analysis (ZBSA), University of Freiburg, 79104 Freiburg, Germany; [email protected] 7 BIOSS Centre for Biological Signalling Studies, University of Freiburg, Schaenzle Str. 18, 79104 Freiburg, Germany 8 Université Côte d’Azur, C3M Inserm U1065, 06204 Nice, France; [email protected] (A.J.); [email protected] (P.C.) 9 INSERM U1065, C3M, Team: Myeloid Malignancies and Multiple Myeloma, 06204 Nice, France 10 Equipe Labellisée par la Fondation ARC, 94803 Villejuif, France 11 Faculty German Cancer Consortium (DKTK), Partner Site Freiburg, Germany and German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany * Correspondence: [email protected] Received: 22 June 2020; Accepted: 19 July 2020; Published: 22 July 2020 Abstract: Background: Cathepsin L (Ctsl) is a cysteine protease mainly located within the endosomal/lysosomal cell compartment. High expression of Ctsl indicates poor prognosis in human breast cancer. However, the cell type-specific Ctsl functions responsible for this association / remain elusive. Methods: Because constitutive Ctsl− − mice develop a complex phenotype, we developed a conditional model allowing for cell type-specific inactivation of Ctsl in mammary epithelium or myeloid cells in the transgenic mouse mammary tumor virus (MMTV)-polyoma middle T (PyMT) breast cancer model. Results: Ctsl ablation in mammary epithelial cells resulted in delayed initiation and end-stage of cancers. The latter displayed large dead cell areas. Inducible in vitro deletion of Ctsl in MMTV-PyMT-derived breast cancer cells revealed expansion of the acidic cell compartment, alteration of intracellular amino acid levels, and impaired mTOR signaling. In consequence, Ctsl-deficient cells exhibited slow growth rates and high apoptosis susceptibility. In contrast to Ctsl-deficient mammary epithelium, selective knockout of Ctsl in myeloid cells had no effects on primary tumors, but promoted lung metastasis formation. Conclusions: Our cell type-specific in vivo analysis provides strong evidence for a cancer cell-intrinsic, tumor-promoting role of Ctsl in primary breast cancer, whereas metastasis is negatively regulated by Ctsl expressed by bone marrow-derived cells. Cancers 2020, 12, 2004; doi:10.3390/cancers12082004 www.mdpi.com/journal/cancers Cancers 2020, 12, 2004 2 of 25 Keywords: breast cancer; lysosome; proteolysis; genetically engineered mice 1. Introduction Proteolysis is a fundamental event at every single stage of tumorigenesis [1,2]. Cysteine cathepsins modulate physiological as well as pathological processes as important components of the intracellular proteolytic network [3]. This protease family is often overexpressed in tumor cells, resulting in their secretion from the acidic cell compartment into the extracellular space [4]. Along with both tumor-promoting and suppressing roles and different cells of origin, cancer model experiments aided the identification of distinct, non-redundant roles of single cysteine proteases in tumor progression. Profiling of single cysteine cathepsins demonstrated their increased activity during defined stages of tumor progression [5]. Some of them, such as cathepsin Z (Ctsz), were found to be upregulated in tumor-associated macrophages (TAMs) following cathepsin B (Ctsb) knockout [6,7]. Cysteine cathepsins released in the tumor microenvironment (TME) promote tumorigenesis in several ways, e.g., by processing different growth factors, cytokines, and chemokines; by cleaving cell-cell junction proteins; or by remodeling the extracellular matrix (ECM) [8,9]. They also play a role in many other tumor-suppressing processes, such as cell death and autophagy, which can contribute to worsening by the development of drug resistance [10]. Although the function of cathepsin L (Ctsl) in the complex process of tumorigenesis is not yet fully understood, the upregulation of its mRNA and protein levels especially in breast cancer correlates with a higher risk of relapse, poor therapy outcome, and worse overall survival [11–13]. Ctsl has been shown to have many unique cell type-specific functions crucial for the maintenance of tissue homeostasis, which cannot be compensated by other cathepsins or other cysteine proteases. By means of Ctsl null mice, the roles of this protease in epidermal homeostasis, hair follicle morphogenesis and cycling, cardiac function, and MHC-II-mediated antigen presentation of cortical thymic epithelial cells were described previously [14–16]. Many other substrates of Ctsl lysosomal activity are arising, together with crucial functions in the development and homeostasis of diverse tissues, e.g., as part of vesicles, Ctsl takes part in the proteolytic processing of neurotransmitters and hormones [17–19]. Due to its tissue-specific functions, the role of Ctsl in several cancer types is versatile. Tumor promoting effects were reported for the RIP1-Tag 2 pancreatic islet cell carcinogenesis model [20]. This report / established a reduction in tumor growth in Ctsl− −animals, resulting from the combination of impaired TAM proliferation and enhanced cell death. A further Ctsl knockout study using the MycER -BclxL pancreatic neuroendocrine cancer revealed an impairment in tumor progression toward the latest stages, an increase in tumor cell death, and elevated expression of autophagy markers, together with defective fusion of autophagosomes and lysosomes [21]. In contrast, several other studies revealed protective functions of Ctsl expression towards carcinogenesis. In a report of intestinal tumorigenesis using the ApcMin model, Ctsl deficiency resulted in an increased tumor incidence as a result of the interplay between Ctsl and the tight-junction protein claudin 1 [22]. Consistently, Ctsl knockout in two squamous cell carcinoma models showed an earlier onset of tumors accompanied by an increase in tumor burden and invasiveness, which was explained by hyper-responsiveness to growth factor signals and hyper-activation of the MAPK/AKT pathways [23,24]. A previous study using the mouse mammary tumor virus (MMTV)-polyoma middle T (PyMT) breast cancer model revealed a massively enhanced metastatic burden in the lungs following transgenic overexpression of human Ctsl [25]. Multiple approaches have been employed to surpass the complex phenotype caused by the lack of Ctsl in mice and, at the same time, to enable the study of the cell type-specific contribution of Ctsl to carcinogenesis. In order to analyze to what extent Ctsl supplied by TAMs contributes to tumorigenesis, / bone marrow from Ctsl− − donor mice was transplanted to RIP1-Tag 2 recipient mice. It could be established that the tumor-promoting functions of Ctsl must be derived from either cancer cells or cells other than from the bone marrow [26]. Additional studies highlighted that restoring the Ctsl catalytic Cancers 2020, 12, 2004 3 of 25 activity in epidermal keratinocytes in a tissue-specific manner can counteract the enhanced malignant / phenotype observed in Ctsl− − skin cancers [23]. Contrary to the aforementioned efforts, the present study reports a direct approach for exploring cell type-specific Ctsl functions in primary cancers by targeting the protease using a Cre/loxP strategy. Conditional Ctsl deletion in mammary epithelium and the cancer cells derived therefrom or, alternatively, in myeloid cells capable of infiltrating breast tumors displayed distinct, tissue-specific functions of Ctsl in the maintenance of cell homeostasis, survival, and proliferation in breast cancer. We further provide evidence for an important intracellular function of Ctsl related to lysosomal homeostasis and lysosome-dependent mTOR signaling. 2. Results 2.1. Generation and Characterization of Conditional Ctsl Knockout Mice We made use of the Cre/loxP technology to address cell-specific functions of Ctsl in murine breast cancer. Ctsl was targeted by flanking exons 3–6 with loxP sites (Figure S1A III). Cre-mediated recombination was predicted to result in the deletion of those exons and in a frameshift-mutation terminating Ctsl translation (Figure S1A IV). As a proof of concept, Ctslfl/fl mice were crossed with Sox2-Cre mice, thereby giving rise to litters bearing a ubiquitous deletion of Ctsl (Figure S1B). Accordingly, Ctsl protein was absent in the kidney and liver of those animals. Furthermore, Ctsl mRNA levels were also undetectable
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